The present invention relates to recombinant DNA molecules encoding plasmid DNA replication origins in Thermus, as well as to shuttle vectors which contain the same.
Many species of bacteria contain small circular extrachromosomal genetic elements, known as plasmids. Plasmids have been found in a number of bacteria which live in extreme environments, including the thermophiles, which live at high temperatures of more than 55xc2x0 C. (Munster et al., Appl. Environ. Microbiol. 50:1325-1327 (1985); Kristjansson and Stetter, in xe2x80x98Thermophilic Bacteriaxe2x80x99, Kristjansson, ed., p. 1-18 (1992)). However, most thermophile plasmids remain xe2x80x98crypticxe2x80x99 in that functional genes have not been isolated from them, hence leaving their functional significance speculative (Hishinuma et al., J. Gen. Microbiol. 104:193-199 (1978); Eberhard et al., Plasmid 6:1-6 (1981); Vxc3xa1squez et al., FEBS Lett. 158:339-342 (1983)). Common genes found in plasmids include those encoding plasmid replication and cellular maintenance, antibiotic resistance, bacteriocin production, sex determination, and other cellular functions (Kornberg and Baker, xe2x80x98DNA Replicationxe2x80x99, 2nd ed. (1991)).
It is often particularly difficult to cultivate thermophilic bacteria within the laboratory. They require high temperatures and often-unknown environmental conditions for acceptable growth (Kristjansson and Stetter, in xe2x80x98Thermophilic Bacteriaxe2x80x99, Kristjansson, ed., p. 1-18 (1992)). However, with the advent of genetic engineering, it is now possible to clone genes from thermophiles into more easily cultivatable laboratory organisms, such as E. coli (Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992)). The expression of such genes can be finely controlled within E. coli. 
A Thermus-E. coli shuttle vector would be desirable if one needs to have the convenience of cloning in E. coli, isolation of DNA from E. coli for further manipulations and subsequently gene selection and expression in Thermus. Such Thermus-E. coli shuttle vectors could be used to screen, select and express thermostable proteins in Thermus. Using these vectors, a gene could, for example, be mutated within a mesophile, transferred to a thermophile, and then its encoded protein selected for increased thermostability. In this way, mesophile-thermophile shuttle-vectors can be used to conduct directed evolution, or protein engineering, on desirable gene products.
There is commercial incentive to produce thermostable proteins which are usually more thermostable in denaturing conditions then mesophilic counterparts (Wiegel and Ljungdahl, CRC Crit. Rev. Biotech. 3:39-108 (1984); Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992)). These thermostable enzymes can also be used in a variety of assays, such as PCR, restriction enzyme-mediated PCR, thermo-cycle DNA sequencing and strand-displacement amplification, in which high temperatures are desirable. The shuttle vectors of the present invention should facilitate production of such thermostable proteins.
The present invention relates to recombinant DNA molecules encoding plasmid DNA replication origins in Thermus, as well as to shuttle vectors which contain the same.
Mesophile-thermophile shuttle vectors require origins of replication (oris) to be genetically maintained and transferred within each bacterial species. To construct appropriate mesophile-thermophile shuttle-vectors, restriction digested thermophile plasmid DNA fragments were ligated into the mesophilic vector pUC19-KmR (the thermostable KmR marker can be selected at 50xc2x0-65xc2x0 C.). Plasmid pUC19 uses the ColEI ori to replicate within E. coli, and does not replicate within the plasmid-accepting thermophile Thermus thermophilus HB27 or HB27 Proxe2x88x92 (Koyama et al., J. Bacteriol. 166:338-340 (1986)). We reasoned that the introduction of plasmid DNA from related Thermus species, which contained a complete thermophilic ori, would confer plasmid replication within HB27.
The thermophilic eubacterium Thermus species YS45 (Raven et al., Nucl. Acids Res. 21:4397 (1993)) contains two cryptic plasmids, and grows between 55xc2x0 C. and 70xc2x0 C. These two Thermus plasmids were named pTsp45S and pTsp45L. These plasmids were digested with a variety of restriction endonucleases to produce fragments that can be cloned into pUC19-derived vectors. A pUC19-derived plasmid with a 4.2-kb XbaI fragment of the small plasmid (pTsp45S, 5.8 kb) of YS45 replicated within HB27. Therefore this XbaI fragment must contain a thermophilic ori. Subsequent deletion analysis revealed that only 2.3 kb (an NheI fragment) within the 4.2 kb was necessary for thermophilic plasmid replication, and that it encodes a replication protein (RepT). The repT gene encodes the 341 amino acid protein, RepT, with predicted molecular mass of 38.2 kDa.
A second Thermus plasmid replication origin from pTsp45L was defined within a 9 kb SphI fragment. This fragment encodes a gene (parA) for plasmid replication and partition. It also contains direct repeats of 5xe2x80x2 RRCTTTTYYY 3xe2x80x2 (SEQ ID NO:1), 5xe2x80x2 RRYTTTG 3xe2x80x2 (SERQ ID NO:2), and an inverted repeat of
5xe2x80x2 TTAACCTTTTTTCAAGAAAAAGAGATAA 3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:3)
3xe2x80x2 AATTGGAAAAAAGTT CTTTTTCTCTATT 5xe2x80x2xe2x80x83xe2x80x83(COMPLEMENT OF SEQ ID NO:3)
The direct repeats and inverted repeats are important for pTsp45L plasmid replication. Deletion of these repeats abolished replication activity in Thermus.